Two (2) important features of the vulnerable plaque are the presence of large necrotic cores, which arise from macrophage death, and inflammation. In addition, advanced lesional macrophages accumulate large amounts of free cholesterol (FC). Using cultured macrophages, we have shown that FC loading induces inflammatory cytokines and activation of p38 MAPK and the unfolded protein response (UPR). This is followed by down-regulation of the anti-apoptotic protein Bcl-2, induction of UPR/p38-mediated apoptosis, and, in the absence of phagocytosis, secondary cellular necrosis. Importantly, markers of the UPR are associated with macrophages in advanced atherosclerotic lesions. In this context, we hypothesize that macrophage apoptosis and inflammatory responses in advanced atherosclerotic lesions, coupled with defective phagocytic clearance of the apoptotic cells, promote progression to the vulnerable plaque. To test this hypothesis, we will explore the role of the MKK3-p38 MAP kinase pathway in cholesterol-induced macrophage death in vitro and in vivo (Aim I); investigate the FC-induced inflammatory response and the consequences of phagocytic clearance of FC-loaded macrophages (Aim II); and determine the role of Bcl-2 in FC-induced macrophage apoptosis and plaque vulnerability (Aim III). Each Aim will consist of a series of mechanistic experiments using cultured macrophages and, to determine relevance to plaque morphology, in vivo experiments using mice with targeted mutations in critical genes in the aforementioned pathways: macrophage p38; the apoptotic cell phagocytic receptor Mer; and macrophage Bcl-2. The ultimate goal is to elucidate how cholesterol-induced apoptosis and inflammatory cytokine production in macrophages and phagocytic clearance of apoptotic cells affect those late lesional characteristics that eventually lead to atherothrombotic vascular occlusion. New insights in this area may suggest new therapeutic strategies to combat cardiovascular disease.